Supplementary Table 1: Sources and sequences of siRNAs. · GE Healthcare Dharmacon, Inc....

Supplementary Table 1: Sources and sequences of siRNAs.

Provider Cat # Target sequence Non‐targeting

siRNA

GE Healthcare Dharmacon, Inc. #D‐001810‐02 UGGUUUACAUGUUGUGUGA

GSK3β siRNA B1 GE Healthcare Dharmacon, Inc. #D‐041080‐02 GAGGAGAGCCCAAUGUUUC

GSK3β siRNA B2 GE Healthcare Dharmacon, Inc. #D‐041080‐03 GCACCAGAGUUGAUCUUUG

BDNF siRNA GE Healthcare Dharmacon, Inc. #D‐042566‐01

#D‐042566‐02 #D‐042566‐03 #D‐042566‐04

UAUGUACACUGACCAUUAA

GAGCGUGUGUGACAGUAUU

GAACUACCCAAUCGUAUGU UCAUAAGGAUAGACACUUC

HDAC1 siRNA GE Healthcare Dharmacon, Inc. #J‐040287‐22

#J‐040287‐23 #J‐040287‐24 #J‐040287‐25

GGGAGAAGGUGGUCGCAAG

ACUAUGGUCUCUACCGAAA UGAACUACCCACUGCGAGA CCAGAACACUAACGAGUAC


#J‐046158‐06 #J‐046158‐07 #J‐046158‐08

CCAAUGAGUUGCCAUAUAA

CAAUUGGGCUGGAGGACUA

ACAGGAGACUUGAGGGAUA

CAAAAGUGAUGGAGAUGUA


#J‐043553‐06 #J‐043553‐08 #J‐043553‐17

GGGAAUGUGUUGAAUAUGU

CGGCAGACCUCCUGACGUA GCACCCGCAUCGAGAAUCA UAUAAGAAGAUGAUCGUCU


#J‐043626‐06 #J‐043626‐07 #J‐043626‐08

GGUUAUGCCUAUCGCAAAU

GUGGAUAGCGACACCAUAU GAAAUUACGCUCAAGGCUU CAACAUGGCUUUCACGGGU

IGF2 siRNA GE Healthcare Dharmacon, Inc. #J‐043709‐09 GGCCAGAUAAGGAGAUCGA

Supplementary Table 2: Primers used for qRT‐PCR analyses

Forward Reverse

Igf2 TGTGCTGCATCGCTGCTTAC CGGTCCGAACAGACAAACTGA

Kcne2 CATCCTGTACCTCATGGTGATG TGGCCTTGGAGTCTTCCAGAT

Sostdc1 TACACCCGTCAGCACAACGA CTCAGACTGTGCTTGCTGGATT

Supplemental Figure 1. Glycogen synthase kinase-3β (GSK3β) immunohistochemistry

We tested if intranasal administration of GSK3β siRNA lowered GSK3β levels in the hippocampus

or perirhinal cortex of wild-type (WT) mice or Fmr1-/- mice. A. Representative staining of GSK3β-

labeled neurons in WT mice treated with scrambled siRNA (n=7) (A) dentate gyrus (DG), (B)

CA3, and (C) CA1, with GSK3β siRNA-B1 (n=5) (D) DG, (E) CA3, and (F) CA1, or with GSK3β

siRNA-B2 (n=6) (G) DG, (H) CA3, and (I) CA1. GSK3β-labeled neurons in Fmr1-/- mice treated

with scrambled siRNA (n=5) (J) DG, (K) CA3, and (L) CA1, treated with GSK3β siRNA-B1 (n=6)

(M) DG, (N) CA3, and (O) CA1, or treated with GSK3β siRNA-B2 (n=5) (P) DG, (Q) CA3, and

(R) CA1. (S) Quantitation of GSK3β-labeled neurons in the hippocampus DG (WT: F(2,17)=3.19,

p<0.05; Fmr1-/-: F(2,15)=7.59, p<0.05), CA3 (WT: F(2,17)=9.17, p<0.01; Fmr1-/-: F(2,15)=6.20,

p<0.05), and CA1 (WT: F(2,17)=8.11, p<0.01; Fmr1-/-: F(2,15)=22.73, p<0.01) in WT and Fmr1-/-

mice. Values are means±SEM. (*p<0.05 compared to scrambled siRNA-treated values in the same

genotype) (cc:corpus callosum; siB1: GSK3β siRNA sequence 1; siB2:GSK3β siRNA sequence

2). Scale bars: 400 µm in all images; dashed lines delineate area of interest.

Representative staining of GSK3β-labeled neurons in perirhinal cortex in wild-type (WT) mice

treated with (T) scrambled siRNA (n=7), (U) GSK3β siRNA-B1(n=5), or (V) GSK3β siRNA-B2

(n=6). GSK3β-labeled neurons in perirhinal cortex in Fmr1-/- mice treated with (W) scrambled

siRNA (n=5), (X) GSK3β siRNA-B1 (n=5), or (Y) GSK3β siRNA-B2 (n=5). (Z) Quantitation of

GSK3β-labeled neurons in the perirhinal cortex (WT: F(2,17)=7.08, p<0.01; Fmr1-/-:

F(2,15)=10.23, p<0.01) in WT and Fmr1-/- mice. Values are means±SEM. (*p<0.05 compared to

scrambled siRNA-treated values in the same genotype) (ec:external capsule; siB1: GSK3β siRNA

sequence 1; siB2:GSK3β siRNA sequence 2). Scale bars: 400 µm in all images. Each symbol

represents the value from an individual mouse.

Supplemental Figure 2. Histone deacetylase-2 (HDAC2) immunohistochemistry

We tested if intranasal administration of HDAC2 siRNA lowered HDAC2 levels in the

hippocampus of wild-type (WT) mice or glycogen synthase kinase-3 knockin (KI) mice.

Representative staining of HDAC2-labeled neurons in the hippocampus. Labeled neurons in wild-

type (WT) mice treated with scrambled siRNA (n=5) (A) dentate gyrus (DG), (B) CA3, and (C)

CA1, or treated with HDAC2 siRNA (n=5) (D) DG, (E) CA3, and (F) CA1. Labeled neurons in

GSK3 knockin (KI) mice treated with scrambled siRNA (n=5) (G) DG, (H) CA3, and (I) CA1, or

treated with HDAC2 siRNA (n=5) (J) DG, (K) CA3, and (L) CA1. (M) Quantitation of HDAC2-

labeled neurons in the hippocampus DG (WT: t(8)=4.34, p<0.01; KI: t(8)=3.54, p<0.01), CA3 (WT:

t(8)=5.28, p<0.01; KI: t(8)=2.51, p<0.05), and CA1 (WT: t(8)=5.58, p<0.01; KI: t(8)=3.15, p<0.05)

in WT and GSK3 KI mice. Values are means±SEM. *p<0.05 compared to scrambled siRNA-

treated values in the same genotype. (cc:corpus callosum) Scale bars: 400 µm in all images; dashed

lines delineate area of interest. Each symbol represents the value from an individual mouse.












treated values in the same genotype (cc:corpus callosum) Scale bars: 400 µm in all images; dashed













treated values in the same genotype (cc:corpus callosum) Scale bars: 400 µm in all images; dashed




hippocampus of wild-type (WT) mice. Representative staining of HDAC4-labeled neurons in the

hippocampus. Labeled neurons in wild-type (WT) mice treated with scrambled siRNA (n=4) (A)

dentate gyrus (DG), (B) CA3, and (C) CA1, or treated with HDAC4 siRNA (n=5) (D) DG, (E)

CA3, and (F) CA1. (G) Quantitation of HDAC4-labeled neurons in the hippocampus DG

(t(7)=4.74, p<0.01), CA3(t(7)=3.50, p<0.05), and CA1 (t(7)=6.62, p<0.01), in WT mice. Values are

means±SEM. *p<0.05 compared to scrambled siRNA-treated values. (cc:corpus callosum) Scale

bars: 400 µm in all images; dashed lines delineate areas of interest. Each symbol represents the

value from an individual mouse.

Supplemental Figure 6. Effects of intranasal treatment with histone deacetylase-4 (HDAC4) or

brain-derived neurotrophic factor (BDNF) siRNA on gene expression.

qRT-PCR was used to measure the hippocampal mRNA levels of Sostdc1, Kcne2 and insulin-like

growth factor-2 (IGF2) in wild-type mice treated intranasally with scrambled siRNA (n=5),

HDAC4 siRNA (n=4), or BDNF siRNA (n=4). Wild-type mice were treated intranasally with

scrambled siRNA or siRNA targeting HDAC4 or BDNF and hippocampal gene expression was

analyzed. (A) Heat map presentation of gene-expression profiles of the genes with significant

differences in expression measured by qRT-PCR (red, high; green, low). HDAC4 and BDNF

siRNA treatments reduced mRNA levels of (B) IGF2 (one-way ANOVA; F(2,12)=5.12, p<0.05)

(*p<0.05 compared to scrambled siRNA-treated WT mice), (C) Kcne2 (one-way ANOVA;

F(2,12)=8.09, p<0.01) (*p<0.01 compared to scrambled siRNA-treated WT mice), and (D) Sostdc1

(one-way ANOVA; F(2,12)=11.44, *p<0.01) (*p<0.01 compared to scrambled siRNA-treated WT

mice). Values are means±SEM. The number of mice (n) for each value is shown within each bar.

Supplemental Figure 7. Cognitive performance of wild-type mice after intranasal insulin-like

growth factor-2 (IGF2) treatment.

We tested if intranasal IGF2 administration altered the performance of wild-type (WT) mice in

novel object recognition, temporal ordering, or coordinate and categorical spatial processing. WT

mice received intranasal vehicle or IGF2 (0.1 μg/mouse/day) 24 hr and 1 hr prior to behavioral

testing (Scheme). (A) WT mice spent significantly more time exploring the novel (N) object than

the familiar (F) object regardless of treatment (*p<0.01) (vehicle: n=8, t(14)=7.49, *p<0.01; IGF2:

n=8, t(14)=4.75, *p<0.01). (B) Discrimination index is shown for novel object recognition

(t(14)=1.15, p=0.27. (C) WT mice spent significantly more time exploring the first object

presented (1) than the most recent object (3) regardless of treatment (*p<0.01) (vehicle: n=8,

t(14)=5.27, *p<0.01; IGF2: n=8, t(14):3.68, *p<0.01). (D) Discrimination index is shown for

temporal ordering (t(14)=0.30, p=0.77). IGF2 treatment of WT mice did not alter (E) coordinate

spatial processing (n=8, t(14)=0.90, p=0.39), or (G) categorical spatial processing (n=8,

t(14)=0.88, p=0.39). Values are means±SEM. Each symbol represents the value from an individual

mouse.

DG

0

20

40

60

80

100

WT Fmr1-/-

*

**

Mice Treatment

WT Scrambled siRNA

WT GSK3 B1 siRNA

WT GSK3 B1 siRNA

Fmr1‐/‐ Scrambled siRNA

Fmr1‐/‐ GSK3 B1 siRNA

Fmr1‐/‐ GSK3 B2 siRNA

Supplemental Figure 1

GSK3

DG

WT Scrambled

DG

WT siB1

DG

WT siB2

CA3

WT Scrambled

CA3

WT siB1

CA3

WT siB2

CA1

cc

WT Scrambled

CA1

cc

WT siB1

CA1

cc

WT siB2

DG

Fmr1‐/‐ Scrambled

DG

Fmr1‐/‐ siB1

DG

Fmr1‐/‐ siB2

CA3


CA3

Fmr1‐/‐ siB1

CA3

Fmr1‐/‐ siB2

cc

CA1


cc

CA1

Fmr1‐/‐ siB1

cc

CA1

Fmr1‐/‐ siB2

400µm A

D

J

G

M

P

O

L

I

F

C

R

B

E

H

K

N

Q

DG CA3 CA1

S

400µm

Fmr1‐/‐ siB1

GS

K3b

po

siti

ve n

euro

ns

Fmr1‐/‐ siB2Fmr1‐/‐Scrambled

WT siB1 WT siB2

WT Scrambled

ec

T

W Y

V U

X

Z

ec

ec ecec

ec


HDAC2


HD

AC

2 p

osi

tive

neu

ron

s

0

50

100

150

200

Scrambled siRNAHDAC2 siRNA

WT GSK3 KI

*

*

WT Scrambled

cc

CA1

WT siRNA HDAC2

cc

CA1

WT Scrambled

CA3

WT siRNA HDAC2

CA3

WT Scrambled

DG

WT siRNA HDAC2

DG

KI Scrambled

cc

CA1

KI siRNA HDAC2

cc

CA1

KI Scrambled

CA3

KI siRNA HDAC2

CA3

KI Scrambled

DG

KI siRNA HDAC2

DG

CA1CA3DG

400µm A

B

D

J

G

L

I

F

CB

E

H

K

M

HDAC1


DG CA1CA3

HD

AC

1 p

osi

tive

neu

ron

s

WT Scrambled

DG

KI Scrambled

DG

CA3

WT Scrambled

CA1

cc

WT Scrambled

CA1

cc

KI Scrambled

DG

WT siRNA HDAC1

CA3

WT siRNA HDAC1

CA1

cc

WT siRNA HDAC1

DG

KI siRNA HDAC1

CA1

cc

KI siRNA HDAC1

CA3

KI siRNA HDAC1

KI Scrambled

CA3

400µm A

D

J

G

L

I

F

CB

E

H

K

M

WT Scrambled

cc

CA1

WT siRNA HDAC3

cc

CA1

WT Scrambled

CA3

WT siRNA HDAC3

CA3

WT Scrambled

DG

WT siRNA HDAC3

DG

KI Scrambled

cc

CA1

KI siRNA HDAC3

cc

CA1

KI Scrambled

CA3

KI siRNA HDAC3

CA3

KI Scrambled

DG

KI siRNA HDAC3

DG

HDAC3


0

20

40

60

80

WT GSK3 KI

*

*

0

20

40

60

80

100

WT GSK3 KI

**

CA1CA3DG

400µm A

B

D

J

G

L

I

F

CB

E

H

K

M

HDAC4


CA3

ScrambledsiRNA

HDAC4siRNA

0

20

40

60

80

100

120

*

DG

ScrambledsiRNA

HDAC4siRNA

0

50

100

150

*

DG

WT Scrambled

DG

WT siRNA HDAC4

CA3

WT Scrambled

CA3

WT siRNA HDAC4

cc

CA1

WT Scrambled

cc

CA1

WT siRNA HDAC4

400µmA B

D

L

F

C

E

G


Ge

ne

Ex

pre

ss

ion

Le

ve

lG

en

e E

xpre

ss

ion

Le

ve

l

Sostdc1

ScrambledsiRNA

HDAC4siRNA

BDNFsiRNA

0.0

0.5

1.0

1.5

2.0*

*

A B

C

D

Sostdc1

Kcne2

IGF2


BC

D E

F G

Temporal order recognition

Novel object recognition

Temporal order recognition

Novel object recognition

Coordinate spatial processing Categorical spatial processing

Vehicle IGF2 -50

0

50

100

Vehicle IGF2 -50

0

50

100

% T

ime

spen

t w

ith

ob

ject

1 3 1 30

20

40

60

80

100

Object:

*

% T

ime

spen

t w

ith

ob

ject

*

Vehicle IGF2

Vehicle IGF20.0

0.2

0.4

0.6

0.8

1.0

Vehicle IGF2 0.5

0.6

0.7

0.8

0.9

1.0

Supplementary Table 1: Sources and sequences of siRNAs. · GE Healthcare Dharmacon, Inc....

Documents

Transcript of Supplementary Table 1: Sources and sequences of siRNAs. · GE Healthcare Dharmacon, Inc....